Protein N-terminal Methylation Mechanisms and Inhibition

蛋白质 N 末端甲基化机制和抑制

• 批准号: 9978827
• 负责人: Rong Huang
• 金额: $ 37.29万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-20 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978827
• 关键词: Aging; Amines; Amino Acid Motifs; Binding; Biochemical; Biochemical Pathway; Bioinformatics; Biological; Biological Assay; Biological Process; Biology; Catalysis; Cell Extracts; Cell physiology; Cells; Cellular Stress; Cellular Stress Response; Chemical Structure; Chemicals; Chromatin; Complex; Coupled; Crystallization; DNA Damage; DNA Repair; Data; Defect; Development; Developmental Process; Disease; Enzymes; Evaluation; Exhibits; Foundations; Functional disorder; Goals; Immunoprecipitation; Kinetics; Knock-out; Knockout Mice; Lead; Link; Malignant Neoplasms; Mediating; Methylation; Methyltransferase; Mitosis; Modification; Molecular; Molecular Structure; Mutation; Mutation Analysis; N-terminal; Pathogenesis; Pathway interactions; Peptides; Pharmacology; Phenotype; Photoaffinity Labels; Physiological; Play; Premature aging syndrome; Property; Protein Methyltransferases; Proteins; Reaction; Reader; Regulation; Research; Roentgen Rays; Role; Route; Series; Specificity; Structure; Structure-Activity Relationship; Time; Western Blotting; Work; analog; cofactor; database structure; demethylation; experimental study; human disease; inhibitor/antagonist; interdisciplinary approach; knock-down; knowledge base; mouse model; mutant; novel; novel therapeutic intervention; protein function; public health relevance; small molecule inhibitor; success; tool

Protein N-terminal methyltransferases (NTMTs) are a relatively new type of protein methyltransferase that catalyze the methylation of alpha-N-terminal amines of proteins starting with an X-P-K/R motif. The alpha-N- terminal methylation plays an essential role in regulating cell mitosis, chromatin interactions, and DNA repair. Its level is increased as response of cellular stress, aging, and developmental processes. Aberrant expression of NTMTs has been observed in various human diseases. However, the biological impact of protein alpha-N- terminal methylation and the molecular basis of NTMT catalysis are poorly defined. Our long-term research goal is to elucidate the biochemical pathways mediated by NTMTs that contribute to the pathogenesis of cancer and developmental defects, and to develop potent and specific NTMT inhibitors. The objective of this research to study mechanism, regulation, and recognition of protein alpha-N-terminal methylation through exploring and applying new chemical biology approaches. Three specific aims will be pursued: Aim 1. Elucidate the molecular and structural basis for substrate and product specificity of NTMTs. We will apply mutational and crystallographic analyses to understand how NTMT1 and 2 recognize their substrates and produce specific methylated products. In addition, we propose to identify a bioorthogonal pair of mutant NTMT1 and SAM analog and use it to obtain a comprehensive profile of physiological substrates for NTMT1. We will carry out biochemical assays and cellular methylation assays to confirm the relevance of identified substrates in cells. Aim 2. Develop potent and specific inhibitors for NTMTs. We will build on our preliminary data to complete synthesis and evaluation of a series of bisubstrate analogs for NTMTs, which will be used as probes to characterize the structural and functional differences of NTMT1 and 2, to investigate NTMT inhibition in cells in comparison with the effects of NTMT1 knockdown. We will extend our crystal structure database of NTMT ternary complexes and to screen for small molecule inhibitors for NTMTs. Aim 3. Identify readers and erasers for protein alpha-N-terminal methylation. There is no information on how this N-terminal methylation is `read' by interacting molecular partners and whether it is static or dynamic (`erasable'). We propose to prepare photoaffinity labeling probes as baits to identify interacting partners of protein alpha-N-terminal methylation in cell extracts. Identified interacting partners will be validated through binding studies and immunoprecipitation experiments. Bioinformatics analysis will be done to develop hypotheses for the biological functions of these interacting partners. Taken together, we believe that this research effort has the great potential to provide a clearer understanding of mechanisms and inhibition of NTMTs, and shed lights on the biological impact of protein N-terminal methylation. Accomplishment of the proposed work will also provide new chemical tools for both basic NTMT biology research and facilitate the development of novel therapeutic approaches to target NTMT1 and NTMT1-involved pathways.

蛋白N末端甲基转移酶（NTMT）是一种相对较新的蛋白甲基转移酶， 催化蛋白质的α-N-末端胺的甲基化，从X-P-K/R基序开始。 alpha-n- 末端甲基化在调节细胞有丝分裂，染色质相互作用和DNA修复方面起着至关重要的作用。 随着细胞应激，衰老和发育过程的反应，其水平被提高。异常表达 在各种人类疾病中已经观察到了NTMT。但是，蛋白α-n-的生物学影响 NTMT催化的末端甲基化和分子基础的定义很差。我们的长期研究 目标是阐明由NTMT介导的生化途径，这有助于有助于的发病机理 癌症和发育缺陷，并发展出有效的NTMT抑制剂。这个目的 研究通过 探索和应用新的化学生物学方法。将追求三个具体目标：目标1。 阐明NTMT的底物和产品特异性的分子和结构基础。我们将申请 突变和晶体学分析以了解NTMT1和2如何识别其底物和 产生特定的甲基化产物。此外，我们建议识别生物正交对突变体 NTMT1和SAM类似物，并使用它来获得NTMT1生理底物的全面概况。 我们将进行生化测定和细胞甲基化测定，以确认已鉴定的相关性 细胞中的底物。目标2。为NTMT开发有效和特异性抑制剂。我们将以我们的初步为基础 数据以完成一系列ntmts的Bisubstrate类似物的综合和评估，将用作 探针表征NTMT1和2的结构和功能差异，以研究NTMT抑制 与NTMT1敲低的作用相比，细胞中的细胞。我们将扩展我们的晶体结构数据库 NTMT三元复合物，并筛选为NTMT的小分子抑制剂。目标3。确定读者和 用于蛋白质α-N-末端甲基化的橡胶。没有关于这种N末端甲基化的信息 通过与分子伙伴进行交互以及它是静态的还是动态的（可擦除”）。我们建议准备 光性标记探针作为诱饵，以识别蛋白质α-N-末端甲基化的相互作用伴侣 细胞提取物。确定的相互作用伙伴将通过结合研究和免疫沉淀来验证 实验。将进行生物信息学分析以开发这些生物学功能的假设 互动合作伙伴。综上所述，我们认为这项研究工作具有提供的巨大潜力 对机制和抑制NTMT的更清晰理解，以及对生物学影响的光明 蛋白N末端甲基化。提议的工作的完成还将为 基本的NTMT生物学研究和促进了新型治疗方法的发展 NTMT1和NTMT1涉及的途径。